RESUMO
Malaria is caused by infection with Plasmodium parasites and results in significant health and economic impacts. Malaria eradication is hampered by parasite resistance to current drugs and the lack of a widely effective vaccine. Compounds that target epigenetic regulatory proteins, such as histone deacetylases (HDACs), may lead to new therapeutic agents with a different mechanism of action, thereby avoiding resistance mechanisms to current antimalarial drugs. The anticancer HDAC inhibitor AR-42, as its racemate (rac-AR-42), and 36 analogues were investigated for in vitro activity against P. falciparum. Rac-AR-42 and selected compounds were assessed for cytotoxicity against human cells, histone hyperacetylation, human HDAC1 inhibition and oral activity in a murine malaria model. Rac-AR-42 was tested for ex vivo asexual and in vitro exoerythrocytic stage activity against P. berghei murine malaria parasites. Rac-AR-42 and 13 achiral analogues were potent inhibitors of asexual intraerythrocytic stage P. falciparum 3D7 growth in vitro (IC50 5-50 nM), with four of these compounds having >50-fold selectivity for P. falciparum versus human cells (selectivity index 56-118). Rac-AR-42 induced in situ hyperacetylation of P. falciparum histone H4, consistent with PfHDAC(s) inhibition. Furthermore, rac-AR-42 potently inhibited P. berghei infected erythrocyte growth ex vivo (IC50 40 nM) and P. berghei exoerythrocytic forms in hepatocytes (IC50 1 nM). Oral administration of rac-AR-42 and two achiral analogues inhibited P. berghei growth in mice, with rac-AR-42 (50 mg/kg/day single dose for four days) curing all infections. These findings demonstrate curative properties for HDAC inhibitors in the oral treatment of experimental mouse malaria.
Assuntos
Antimaláricos , Malária , Parasitos , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Malária/tratamento farmacológico , Camundongos , Plasmodium berghei , Plasmodium falciparumRESUMO
In this work we aimed to develop parasite-selective histone deacetylase inhibitors (HDAC) inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis, and biological testing of a series of 13 terephthalic acid-based HDAC inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC50 : 8->51â µm), with 11 also having sub-micromolar inâ vitro activity against drug-sensitive (3D7) and multidrug-resistant (Dd2) asexual blood-stage P.â falciparum parasites (IC50 ≈0.1-0.5â µm). A subset of compounds were examined for activity against early- and late-stage P.â falciparum gametocytes and P.â berghei exo-erythrocytic-stage parasites. While only moderate activity was observed against gametocytes (IC50 >2â µm), the most active compound (N1 -((3,5-dimethylbenzyl)oxy)-N4 -hydroxyterephthalamide, 1 f) showed sub-micromolar activity against P.â berghei exo-erythrocytic stages (IC50 0.18â µm) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual-stage inâ vitro potency (IC50 ≈0.1â µm) and selectivity of this compound versus human cells (SI>450), suggests that 1 f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Desenho de Fármacos , Ácidos Ftálicos/química , Ácidos Ftálicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Concentração Inibidora 50 , Estágios do Ciclo de Vida/efeitos dos fármacos , Ácidos Ftálicos/síntese química , Relação Estrutura-AtividadeRESUMO
Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 µM) or S. mansoni schistosomula (IC50 > 10 µM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 â¼10 µM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Leishmania/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Acetilação , Administração Oral , Animais , Depsipeptídeos/farmacologia , Células HEK293 , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Concentração Inibidora 50 , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Panobinostat , Parasitemia/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium knowlesi/crescimento & desenvolvimento , Schistosoma mansoni/crescimento & desenvolvimento , Sulfonamidas/farmacologia , VorinostatRESUMO
Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.
Assuntos
Doxorrubicina/farmacologia , Neoplasias Mamárias Experimentais/terapia , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/administração & dosagem , Receptores Proteína Tirosina Quinases/genética , Animais , Apatitas/administração & dosagem , Linhagem Celular Tumoral , Cisplatino/farmacologia , Sinergismo Farmacológico , Feminino , Técnicas de Silenciamento de Genes , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Distribuição AleatóriaRESUMO
BACKGROUND: Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs. METHODS AND RESULTS: We report that carbonate apatite-mediated delivery of the siRNAs targeting ABCG2 and ABCB1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose-dependently enhanced chemosensitivity to doxorubicin, paclitaxel and cisplatin, the traditionally used chemotherapeutic agents. Moreover, codelivery of two specific siRNAs targeting ABCB1 and ABCG2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells, indicating the reversal of ABC transporter-mediated multidrug resistance. CONCLUSION: The delivery concept of multiple siRNAs against ABC transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Inativação Gênica/efeitos dos fármacos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apatitas/farmacocinética , Apatitas/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Terapia Combinada , Relação Dose-Resposta a Droga , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células MCF-7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinéticaRESUMO
Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. RNA interference (RNAi), a powerful tool in functional genomics to selectively silence messenger RNA (mRNA) expression, can be harnessed to rapidly develop novel drugs against any disease target. The ability of synthetic small interfering RNA (siRNA) to effectively silence genes in vitro and in vivo, has made them particularly well suited as a drug therapeutic. However, since naked siRNA is unable to passively diffuse through cellular membranes, delivery of siRNA remains the major hurdle to fully exploit the potential of siRNA technology. Here pH-sensitive carbonate apatite has been developed to efficiently deliver siRNA into the mammalian cells by virtue of its high affinity interactions with the siRNA and the desirable size of the resulting siRNA/apatite complex for effective cellular endocytosis. Moreover, following internalization by cells, siRNA was found to be escaped from the endosomes in a time-dependent manner and finally, more efficiently silenced reporter genes at a low dose than commercially available lipofectamine. Knockdown of cyclin B1 gene with only 10nM of siRNA delivered by carbonate apatite resulted in the significant death of cancer cells, suggesting that the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy.